1. Field of Invention
This invention concerns novel photosensitizers related to rhodoporphyrins and phylloerythrin methyl ester useful for photodynamic therapy. In particular, this invention concerns a variety of rhodoporphyrins, rhodochlorins, mesorhodochlorins, phylloerythrin derivatives, and their preparation and use as photosensitizers for detection and treatment of tumors by photodynamic therapy. The novel photosensitizers accumulate and concentrate preferentially in tumor tissue, have strong light absorption in the 650 to 900 nm region and have reduced skin phototoxicity.
2. Background and Related Art
In the last decade, a photodynamic therapy (PDT) appeared as a new and viable approach to cancer diagnosis and therapy. Photodynamic therapy involves systemic administration of a photosensitizer, a photosensitive compound which in itself is essentially therapeutically inactive but which accumulates preferentially in tumor tissue and absorbs light at wavelength between 660 and 880 mm. The administration of the photosensitizer is followed by illumination with visible light. The lesion or tumor is detected by a presence of the accumulated photosensitizer with visible light. The treatment of tumors by photodynamic therapy, therefore, requires photosensitizers which accumulate in tumor tissue, which are distinguished by large quantum yield to sensitize the formation of singlet oxygen, and which have strong absorption in the spectral region where tissue transmits best, that is between 660 to 800 nm.
Photodynamic therapy (PDT) has been first described in Cancer Res., 38:2628 (1976) and since then it has been used experimentally in cancer patients with an estimated 3,000-4,000 patients treated world-wide. PDT is based on findings published in J. Natl. Cancer Inst., 26:1 (1961) that a derivatized hematoporphyrin (HpD) which has fluorescent properties is selectively uptaken by malignant tissue. Subsequently, studies described in Ann. Surg., 167:829 (1968) indicated that photophyrin hematoporphyrin derivative (HpD) is taken up by a wide variety of carcinomas and sarcomas both in man and animals. The actual therapeutic potential of HpD for treatment of a metastatic breast cancer with light was first disclosed in Proc. IX Int. Cancer Congress, 393 (1966) which describes a positive tumor response to this treatment.
In 1975, J. Natl. Cancer Inst., 55:115 (1975) reported that a transplanted mammary tumor in mice could be eradicated completely using filtered red light following systemic injection of HpD and about the same time, a positive tumor response to a treatment of a bladder tumor was described in a patient injected with HpD by delivering light endoscopically via a glass light guide. In response to the treatment, the tumor size decreased in the treated area with no apparent effect in the untreated area. These findings were reported in J. Urol., 155:150 (1976). Since that time, numerous clinical trials using HpD have been reported encompassing essentially all solid tumors accessible on the skin surface or endoscopically.
So far, several groups of compounds are known to act as photosensitizers. HpD, available as Photofrin I and its more purified version Photofrin II are the first clinically approved photosensitizers developed by Quadra Logic Technologies and Lederle Laboratories. They are prepared by acid catalyzed acetylation of hematoporphyrin (Hp) and subsequent alkaline treatment. These compounds are complex oligomeric mixtures contaminated with starting hematoporphyrin and its dehydration products. The oligomeric mixtures which comprise 50 to 80% of the tumor-localizing fraction of HpD are selectively retained in the tumors and are responsible for both the in vivo fluorescence and for photosensitizing properties of HpD.
In spite of the potential broad diagnostic and therapeutic application of PDT in clinical oncology, there are disadvantages connected with use of HpD because of the lack of a complete understanding of which active component or components in the HpD complex are responsible for tumor uptake and retention. Moreover, the limited tumor selectivity of HpD, the limited tissue penetration of the light upon treatment following HpD administration by systemic injection and limited retention by malignant tissues were observed.
Photodynamic treatment with porphyrins is further complicated by observation that normal nontumorous organs such as liver, kidney, spleen and skin tend to retain considerable amounts of the porphyrins. The porphyrins accumulated in these nontumorigenic tissues react, upon light illumination, exactly as the porphyrins in the tumor tissue, that is by producing cytotoxic singlet oxygen which assert a cytotoxic activity on the normal tissue, thereby causing severe tissue damage and other undesirable side effects. Another problem associated with this treatment is a skin photosensitivity. In order to avoid cutaneous phototoxicity following the treatment with a photosensitizer, a patient must remain in subdued light for four to six weeks after HpD administration.
The other major drawback of HpD is its weak absorbance at 630 nm, the wavelength of red light most commonly used in PDT. Incomplete responses to the HpD treatment in some cases were attributed to the difficulty in delivering light to some tumor sites and to incomplete light penetration for larger tumors due to its weak absorbance.
These problems have resulted in considerable efforts devoted to developing new photosensitizers having increased absorption maxima in the 600-800 nm region in order to increase the efficiency of the light as well as to achieve greater tissue penetration with the longer wavelengths.
Several new classes of photosensitizers for use in PDT are derived from tetrapyroles and their derivatives. Tetrapyrolic macrocycles are the most ubiquitous of all naturally occurring pigments and most of them generate high quantum yields of triplet states from which energy transfer to ground state oxygen appears to be the most dominant process. Synthetic etiopurpurin, benzochlorines, rhodins, verdins, and methyl pyrroverdin as well as the novel pentapyrolic macrocycles pentaphyrin and sapphyrin have shown very promising results in PDT, and are considered to be a new generation of PDT photosensitizers. While these compounds seem to possess absorption wavelength maxima around 750-800 nm, some of these compounds are highly unstable and thus not overly suitable and practical for diagnostic and therapeutic purposes.
Tetrapyrrole compounds and a process for the production of thereof useful in photodiagnosis and phototherapy is described in the EPO patent application 85108981.3 filed on Jul. 18, 1985.
Phthalocyanines and their derivatives are another class of new photosensitizers under investigation. They are structurally related to the naturally occurring porphyrins but have the four isoindole units linked by aza-nitrogens rather than methane linkages. These compounds show strong absorption in the 650-700 nm range, and certain non-metallo as well as metallo derivatives exhibit efficient photochemical processes. However, since the most studied sulfonated phthalocyanines are composed of mono-, di-, tri- and tetrasulfonated species, they may not be the most suitable for human use.
So far the most promising new second generation photosensitizer closest to the clinic is mono-L-aspartylchlorin E.sub.6 (MACE) and a related compound di-L-aspartyl-chlorin-e.sub.6 (DACE) which possess strong absorption bands with high molar extinction coefficients around 664 nm. MACE and DACE are prepared by alkaline degradation from methyl pheophorbide-a followed by esterification with diazomethane to give chlorin E.sub.6 trimethyl ester, which is then converted to the acid chloride. Aspartic acid is then added using usual peptide chemistry.
Initial PDT experiments demonstrated that MACE was ineffective at inducing tumor cures when a 24 hours time interval between drug administration and light treatment was used. However, when MACE was administered 4-6 hours prior to light exposure, PDT induced tumor cures were obtained. In addition, at comparable drug and light doses, the level of PDT induced normal skin damage was significantly lesser for MACE than for HpD. The results of testing indicated that MACE is a short-acting but not overly effective tumor photosensitizer with good in vivo clearance properties. The characterization of MACE and DACE is described in J. Natl. Cancer Inst., 80:330 (1988).
Purpurin-18 and chlorin-p.sub.6, two derivatives of chlorophyll, described in Photochemistry and Photobiology, 48:579 (1988) were found to be potent photosensitizers which promote cell killing by low intensity of red light
Recently, a new class of compounds, namely chlorophyll-a derivatives were found to be very potent photosensitizers. These compounds are disclosed in the copending patent application Ser. No. 07/840,347 filed on Feb. 24, 1992, now U.S. Pat. No. 5,330,741, which is hereby incorporated by reference in its entirety.
The porphyrin and chlorin derivatives which have led to the development of the current invention have been reviewed in Proc. SPIT, 1065:164 (1989). The aspartyl derivatives of chlorin E.sub.6, monoaspartyl chlorin E.sub.6, and diaspartyl chlorin E.sub.6 were found to be effective photosensitizers in vitro. With these compounds, the aspartyl group was noted to be responsible for the efficiency of tissue clearance. In pheophorbide and pyropheophorbide and chlorin E.sub.6 series, certain alkyl ether derivatives, including 2-(1-hexyloxyethyl)2-des vinyl derivatives, were found to be excellent photosensitizers compared with their parent compounds, methyl pyropheophorbide-a, pyropheophorbide-a and chlorine E.sub.6. Chlorin-e.sub.6 esters were found to be slightly less effective than methyl pheophorbide-a and pyrophorbide-a analogs, indicating that either the E-ring is an important factor in photoactivity or that due to its increase in hydrophility, E-ring is not retained in the tumor cells for longer time than the n-hexyl or n-heptyl ether derivatives of pyropheophorbide-a. Among recently newly discovered photosensitizers, some benzoporphyrin derivatives (BPD), usually as a mixture of isomers, have also been reported in J. Natl. Cancer Inst., 79:1327 (1987).
In recent years there has been increased interest in synthesis of long wavelength photosensitizers for use in photodynamic therapy which would result in deeper tissue penetration and allow treatment of larger tumors, particularly, if these new photosensitizers would have reduced skin phototoxicity.
Some attempts to provide such photosensitizers were recently described. However, these attempts were only partially successful. The synthesis of benzoporphyrin derivatives (BPD) from protoporphyrin IX dimethylester according to method described in J. Natl. Cancer Inst., 79:1327 (1987) lacks regiochemical control resulting in a mixture of active and inactive compounds requiring extensive purification before biological studies of these compounds could be done. These compounds showed good vitro photosensitizing efficacy. However, in vivo these compounds were effective only when the animals were treated 3 hours post i.v. injection of the drug. When the treatment was done 24 hours post injection of the drug, even at higher doses (5 mg/kg), no tumor response was observed. These compounds therefore seem to have little tumor selectivity.
A recent methodological advancement described in Bioorg. Med. Chem. Letr., 2:1575 (1992) reduces the separation problems observed above. The method uses 4-acetyl-2-vinyldeuteroporphyrin IX dimethyl ester as a starting material. Since only one vinyl group is present, the additional purification step after Diels-Alder coupling is unnecessary. Although this method has advantages over using the divinylporphyrin, it still requires the synthesis and separation of the two hydroxyethyl isomers obtained from partial reduction of diacetyldeuteroporphyrin IX dimethyl ester.
This invention, consequently, provides a variety of benzoporphyrin derivatives derived from rhodoporphyrins and phylloerythrin methyl ester related photosensitizers. These new photosensitizers are stable compounds which are easy to synthesize, which are able to penetrate deeper in the tissue and therefore allow treatment of larger tumors. The new photosensitizers have substantially reduced skin phototoxicity compared to Photofrine.
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.